KR102108600B1 - Modified tgf-beta2 oligonucleotides - Google Patents

Abstract

The present invention includes modified nucleotides, such as LNA, ENA, polyalkylene oxide-, 2'-fluoro, 2'-O-methoxy and / or 2'-O-methyl modified nucleotides, TGF -Beta2 nucleic acid sequence is directed to an oligonucleotide consisting of 10 to 18 nucleotides of a selected region. The present invention also relates to pharmaceutical compositions comprising such oligonucleotides, wherein the composition or the oligonucleotides are malignant and / or benign tumors, immunological diseases, fibrosis, or ophthalmic diseases such as dry eye, It is used for the prevention and / or treatment of glaucoma or posterior capsule turbidity (PCO).

Description

Modified TGF-beta2 oligonucleotide {MODIFIED TGF-BETA2 OLIGONUCLEOTIDES}

The present invention relates to oligonucleotides consisting of 10 to 18 nucleotides capable of hybridizing with TGF-beta2 nucleic acid sequence, TGF-beta1 or TGF-beta3 nucleic acid sequence, wherein the oligonucleotide is a modified nucleotide, eg For example LNA, ENA, polyalkylene oxide-, 2'-fluoro, 2'-O-methoxy and / or 2'-O-methyl modified nucleotides.

The transforming growth factor beta (TGF-beta) is a protein that regulates proliferation, cell differentiation, and other functions in most cells. The TGF-beta is, among other things, a type of cytokine that plays a role in immunity, cancer, heart disease, diabetes, Marfan's syndrome, Roy-Diet's syndrome, Parkinson's disease, and AIDS.

TGF-beta is a secreted protein that is encoded by different genes but exists in at least three isoforms (TGF-beta1, TGF-beta2 and TGF-beta3) that share strong sequence and structural homology. TGF-beta acts as a proliferation inhibitor in normal epithelial cells and early stages of carcinogenesis. However, at the end of tumor development, TGF-beta can become tumor-promoting through mechanisms involving the induction of epithelial to mesenchymal transition (EMT), a process thought to contribute to tumor progression, invasion and metastasis. See, "Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF) -beta induced epithelial to mesenchymal transition" 7th space. Com.2009-01-08. Retrieved: 2009-01-29 ).

In normal (epithelial) cells, TGF-beta stops the cell cycle (and stops cell proliferation) at the G1 stage, induces differentiation, or promotes apoptosis. When cells are transformed into cancer cells, TGF-beta no longer inhibits cell proliferation (which is often the result of mutations in the signaling pathway), and cancer cells proliferate.

Proliferation of stromal fibroblasts is also induced by TGF-beta. All of these cells increase the production of TGF-beta. The TGF-beta acts on peripheral stromal cells, immune cells, endothelial, smooth muscle cells, and tumor microenvironments (Pickupet al., "The roles of TGF6 in the tumour microenvironment", Nature Reviews Cancer (2013), 13 : 788-799). Thereby, the TGF-beta promotes angiogenesis, and suppresses proliferation and activation of immune cells, thereby causing immunosuppression.

TGF-beta1-deficient mice die from inflammation of the heart, lungs and stomach, suggesting that TGF-beta plays an extremely important role in inhibiting the activation and proliferation of inflammatory cells. Smad3 is one of the key elements in the TGF-beta dependent downstream signaling pathway. Smad3-deficient mice exhibit chronic mucosal infections due to T-cell activation and impaired mucosal immunity, suggesting a key role for TGF-beta in this process. With respect to cancer, the production and secretion of TGF-beta by some cancer cells inhibits the activity of infiltrating immune cells, helping the tumor to escape host immunosurveillance. This immunosuppressive effect may be another important mechanism by which TGF-beta stimulates the growth of terminal tumors (Blobe GC et al., May 2000, "Role of transforming growth factor beta in human disease", N. Engl J. Med. 342 (18), 1350-1358). TGF-beta also converts effector T-cells, which typically attack cancer with an inflammatory (immune) response, into regulatory (inhibitory) T-cells that turn off the inflammatory response.

Moreover, TGF-beta is one of the most potent modulators of the production and deposition of extracellular matrix. The TGF-beta stimulates the production of the extracellular matrix by two main mechanisms and affects the adhesion properties. First, TGF-beta stimulates fibroblasts and other cells to produce extracellular-substrate proteins and cell-attached proteins (including collagen, fibronectin and integrin). Second, TGF-beta reduces the production of enzymes that break down the extracellular matrix (including collagenase, heparinase, and stromelysin), and inhibits the enzymes that break down the extracellular matrix (type 1) Production of plasminogen-activator inhibitors and metalloprotease tissue inhibitors). The net effect of this change is to increase the production of extracellular-substrate proteins and increase or decrease the adhesion properties of cells in a cell-specific manner. The production of TGF-beta in many cancer cells increases, which increases the proteolytic activity of the TGF-beta and increases its invasiveness by promoting its binding to cell-adhesive molecules (Blobe GC et al. ., May 2000, "Role of transforming growth factor beta in human disease", N. Engl. J. Med. 342 (18), 1350-1358).

Thus, therapeutic agents that can each affect TGF-beta expression and activity are essential, particularly for use in the prevention and / or treatment of TGF-beta related diseases. EP 1008649 and EP 0695354, for example, are oligonucleotides suitable for hybridization with mRNAs of TGF-beta1 and / or TGF-beta2 and for use in the manufacture of pharmaceutical compositions for the prevention and / or treatment of cancer, for example. Disclosed. None of these oligonucleotides contain modifications such as LNA, ENA, and the like.

WO 2003/85110, WO 2005/061710, and WO 2008/138904 are directed to oligonucleotides, which may include, for example, modifications of nucleotides to the inhibition of HIF-1A, Bcl-2 and HER3, respectively, and may be useful in the treatment of cancer. will be.

The selection criteria for oligonucleotides are mainly the length of the oligonucleotide, GC-percentage, hairpin formation tendency, dimerization, and melting temperature (Tm). In general, a high Tm (melting temperature) is preferred. Moreover, the oligonucleotide must be specific to the target mRNA to reduce potential off-target effects and will not hybridize to the non-target mRNA.

Therefore, there is a great scientific and medical need for therapeutic agents that reduce or inhibit TGF-beta expression and / or activity. In particular, as well as antisense oligonucleotides that specifically interact and thus reduce or inhibit the expression of TGF-beta1, TGF-beta2, and / or TGF-beta3, without causing any (severe) side effects. Oligonucleotides, such as oligonucleotides that specifically inhibit TGF-beta1 and TGF-beta2, or TGF-beta1 and TGF-beta3, or TGF-beta2 and TGF-beta3, have long been required.

The present invention relates to oligonucleotides consisting of 10 to 18 nucleotides of the TGF-beta2 nucleic acid sequence of SEQ ID NO: 1 (see FIG. 1), wherein one or more nucleotide (s) of the oligonucleotide is modified. Preferred oligonucleotides comprising or consisting of one of SEQ ID NOs: 2 to 20 are shown in Table 1. These oligonucleotides are very effective in reducing and inhibiting TGF-beta2 expression and activity, respectively.

Preferred oligonucleotides of the present invention are ASPH47, ASPH190, ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197, ASPH198, ASPH199, ASPH200, ASPH201, and ASPH202, ASPH203, ASPH204, ASPH205, ASPH206, ASPH207, ASPH208, respectively. , ASPH210, ASPH211, ASPH212, ASPH213, ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221, ASPH222, and ASPH223.

Modifications of one or more of the oligonucleotides of the present invention include LNA, ENA, polyalkylene oxides such as triethylene glycol (TEG), 2'-fluoro, 2'-O-methoxy and 2'-O-. It is selected from the group consisting of methyl. The modification is preferably located at the 5'- and / or 3'-end of the oligonucleotide. Oligonucleotides comprising such modified nucleotides are modified oligonucleotides.

The modified nucleotides are arranged, for example, in a line, one next to the other, or in a different pattern (where one or more unmodified nucleotides follows the modified nucleotide). For example, an oligonucleotide starts with one or more modified nucleotides, followed by one or more, such as 1, 2, 3 or 4 unmodified or unlocked nucleotides, followed by one or more modified nucleotides. . In one embodiment, both ends of the oligonucleotide include modified and unmodified or unlocked nucleotides of the same pattern. In another embodiment, the modification patterns at the 3'- and 5'-ends are different, including that one end does not contain modified nucleotides. Preferably, the modified oligonucleotide comprises a series of 8 or 9 unlocked nucleotides.

On the one hand, the nucleotide is modified at any other position in the oligonucleotide, or at least one nucleotide is modified at the 5'- and / or 3'-end of the oligonucleotide and at any other position in the oligonucleotide. The oligonucleotide includes one type of modification, or one or more different modifications. Optionally, the at least one phosphate linkage between two consecutive nucleotides of the oligonucleotide (modified or unmodified) is phosphorothioate or methylphosphonate. In a preferred embodiment, the oligonucleotide of the invention is phosphorothioate.

The oligonucleotides of these different embodiments are all malignant or benign tumors, immunological diseases, fibrosis (e.g. idiopathic pulmonary fibrosis, renal fibrosis, renal fibrosis), sclerosis (e.g. cirrhosis of the liver), scleroderma or related skin diseases, eye diseases, For example, for use in prevention and / or treatment methods such as glaucoma or posterior capsule turbidity (PCO), CNS disease, and hair loss.

1 shows the nucleic acid sequence of human TGF-beta2 mRNA (NM_003238.3).
2 shows examples of nucleotide modifications.
Figure 3 shows the inhibition of the expression of TGF-beta1, TGF-beta2 and TGF-beta3 mRNA in human Panc-1 pancreatic cancer cells and mouse RenCa renal cell carcinoma cells. Panc-1 cells and RenCa cells were treated with a 1.1 μM dose of different modified oligonucleotides in the absence of any transfection reagents (gymnotic transfection or unsupported transfection or gynotic delivery) and TGF-beta Inhibition of 1 (black column), TGF-beta 2 (white column) and TGF-beta 3 (comb column) mRNA expression was measured after 72 hours. Figure 3 is a modified oligonucleotide ASPH190, ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197, ASPH198, ASPH199, ASPH200, ASPH201, ASPH202, ASPH203, ASPH204, ASPH205, ASPH206, ASPH207, ASPH208, ASPH209, ASPH207, respectively The results are for ASPH211, ASPH212, ASPH213, ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221, ASPH222, and ASPH223. Figure 3a shows the inhibitory effect of these TGF-beta oligonucleotides in Panc-1 cells and Figure 3b shows the effect in RenCa cells.
4 shows the inhibitory effect of the oligonucleotide of the present invention on the expression of TGF-beta1 and TGF-beta2 proteins. Human Panc-1 cells were transfected with 20, 6.67, 2.22, 0.74, 0.25, 0.08 or 0.009 μM of the modified oligonucleotide ASPH47 (FIG. 4A). The negative control is the scrambled oligonucleotide (scrLNA) of SEQ ID NO: 22 at concentrations of 40, 13.33, 4.44, 1.48, 0.49, 0.16, 0.05, or 0.02 μM (FIG. 4B). TGF-beta1 (diamond shape) and TGF-beta2 (square) protein levels in cell supernatant were measured by ELISA.

The present invention relates to oligonucleotides, particularly antisense oligonucleotides, comprising at least one modified nucleotide and suitable for interaction with TGF-beta mRNA, preferably TGF-beta1, TGF-beta2 and / or TGF-beta3 will be. The oligonucleotide comprises or consists of 10 to 18 nucleotides of the TGF-beta2 nucleic acid according to SEQ ID NO: 1. Most preferably, the oligonucleotide comprises or consists of 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides. The oligonucleotide is a single or double stranded RNA or DNA, including siRNA, microRNA, aptamer or spiegelmer. Preferably, the oligonucleotide is an antisense oligonucleotide.

Preferred oligonucleotides of the present invention are ASPH47, ASPH190, ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197, ASPH198, ASPH199, ASPH200, ASPH201, and ASPH202, ASPH203, ASPH204, ASPH205, ASPH206, ASPH207, ASPH208, respectively. , ASPH210, ASPH211, ASPH212, ASPH213, ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221, ASPH222, and ASPH223. Antisense oligonucleotides of the present invention may be disclosed differently, for example ASPH47, ASPH0047, ASPH_47 or ASPH_0047 refers to the same oligonucleotide.

The nucleotides form the building blocks of oligonucleotides, e.g. nucleobases (nitrogen bases, e.g. purines or pyrimidines), 5-carbon sugars (e.g. ribose, 2-deoxyribose, arabinose, xylose, Lyxos, allos, altos, glucose, mannose, gulose, idos, galactose, talos or stabilized modifications of these sugars), and one or more phosphate groups. Examples of modified phosphate groups are phosphothioate or methylphosphonate. Each compound of the nucleotides is deformable, natural or unnatural. The non-natural compound is, for example, locked nucleic acid (LNA), 2'-O, 4'-C-ethylene-crosslinked nucleic acid (ENA), polyalkylene oxide- (for example triethylene glycol (TEG)), 2 '-Fluoro, 2'-O-methoxy and 2'-O-methyl modified nucleotides (eg Freier & Altmann, Nucl. Acid Res., 1997, 25, 4429-4443) and Uhlmann , Curr.Opinion in Drug & Development (2000, 3 (2): 293-213)) (these are shown in Figure 2).

LNA is a modified RNA nucleotide, wherein the ribose moiety is modified with an extra bridge linking 2 'oxygen and 4' carbon (2'-4 'ribonucleoside). The bridge "locks" the ribose into a 3'-endo (north) configuration, often found in type A duplexes. LNA nucleosides and nucleotides each include, for example, the form of thio-LNA, oxy-LNA, or amino-LNA in the alpha-D- or beta-L-configuration, respectively, with DNA or RNA residues in the oligonucleotide, respectively. It is mixable and combinable.

The oligonucleotides of the present invention, i.e., modified oligonucleotides, comprise one or more modified nucleotides, preferably LNA and / or ENA, at the 5'- and / or 3'-end of the oligonucleotide. In a preferred embodiment, the oligonucleotide comprises 1, 2, 3 or 4 LNA or ENA at the 5'-end, and 1, 2, 3 or 4 LNA or ENA at the 3'-end. In another preferred embodiment, the oligonucleotide is 1, 2, 3 or 4 LNA or ENA at the 5'-end or 3'-end, and a polyalkylene oxide at the 3'- or 5'-end, e.g. For include TEG. The modified oligonucleotides each exhibit a markedly increased inhibition of TGF-beta expression and activity, resulting in malignant or benign tumors, immunological diseases, fibrosis, eye diseases such as dry eye, glaucoma or posterior capsule turbidity (PCO). ), And improved prevention and / or treatment of CNS disease, hair loss, and the like. The oligonucleotides of the present invention target TGF-beta related diseases by hybridization with TGF-beta mRNA, preferably TGF-beta1, TGF-beta2, or TGF-beta3.

Preferably two or more oligonucleotides are used in combination, wherein one or more oligonucleotides specifically inhibit TGF-beta1 and one or more oligonucleotides specifically inhibit TGF-beta2, or one or more oligonucleotides Specifically inhibit TGF-beta1 and one or more oligonucleotides specifically inhibit TGF-beta3, or one or more oligonucleotides specifically inhibit TGF-beta2 and one or more oligonucleotides TGF-beta 3 specifically, or one or more oligonucleotides specifically inhibit TGF-beta1, one or more oligonucleotides specifically inhibit TGF-beta2, and one or more oligonucleotides TGF-beta3 Specifically suppressed. The oligonucleotides of the present invention most preferably inhibit the expression and / or activity of TGF-beta2 mRNA.

In another embodiment, one oligonucleotide has two TGF-beta isoforms, such as TGF-beta1 and TGF-beta2, TGF-beta2 and TGF-beta3, or TGF-beta1 and TGF- Inhibit beta3. Oligonucleotides that inhibit the expression of two or three isoforms of TGF-beta1, TGF-beta2 and TGF-beta3 are defined as pan-specific oligonucleotides.

In a further embodiment, three or more oligonucleotides are used in combination, wherein one or more oligonucleotides specifically inhibit TGF-beta1, and another oligonucleotide specifically inhibits TGF-beta2, further The oligonucleotide of specifically inhibits TGF-beta3, and optionally one or more additional oligonucleotides inhibit TGF-beta1, TGF-beta2 or TGF-beta3.

The oligonucleotides of the invention have an IC ₅₀ in the range of, for example, 0.1 to 20 μM, preferably in the range of 0.2 to 15 μM, more preferably in the range of 0.4 to 10 μM, and even more preferably in the range of 0.5 to 5 μM. .

The invention also relates to a pharmaceutical composition comprising an oligonucleotide according to the invention as an active ingredient. The pharmaceutical composition comprises one or more oligonucleotides of the invention and optionally further antisense compounds, antibodies, chemotherapy compounds, anti-inflammatory compounds, antiviral compounds and / or immuno-modulating compounds. Pharmaceutically acceptable binders and adjuvants are optionally included by the pharmaceutical composition.

In one embodiment, the oligonucleotide and pharmaceutical composition are formulated as a dosage unit in the form of a solution, each containing a binder, excipient, stabilizer, and the like.

The oligonucleotide and / or pharmaceutical composition can be administered via different routes. These routes of administration include, but are not limited to, electroporation, epidermis, imprinting into the skin, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrathecal, intra-abdominal, intraperitoneal, and prostate. , Intrapulmonary, intrathecal, intratracheal, intratumoral, intravenous, bladder, intranasal placement, nasal inhalation, oral, pulmonary inhalation (e.g. by inhalation or inhalation of powders or aerosols, including nebulizers), subcutaneous , Subcutaneous, topical (including mucosa including eye and vaginal and rectal delivery), or transdermal.

For parenteral, subcutaneous, transdermal or topical administration, the oligonucleotide and / or pharmaceutical composition comprises, for example, sterile diluents, buffers, toxicity modifiers and antibacterial agents. In a preferred embodiment, the oligonucleotide or pharmaceutical composition is prepared from a carrier that protects against degradation or immediate removal from the body, for example, implants or microcapsules with controlled release properties. For intravenous administration, preferred carriers are, for example, physiological saline or phosphate buffered saline. For oral administration, oligonucleotides and / or compositions comprising such oligonucleotides may be, for example, powders or granules, particulates, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or Includes mini tablets. Oligonucleotides and / or pharmaceutical compositions for parenteral, intrathecal, anterior or intraventricular administration may include, for example, buffers, diluents and other suitable additives such as penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. It includes a sterile aqueous solution optionally containing.

The pharmaceutically acceptable carrier is, for example, liquid or solid, and is selected in a planned administration manner in consideration of providing a desired bulk, consistency, etc. when used in combination with a nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binders (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); Fillers (eg lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or calcium hydrogen phosphate, etc.); Lubricants (eg magnesium stearate, talcum, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); Disintegrants (eg starch, sodium starch glycolate, etc.); Or wetting agents (eg sodium lauryl sulfate, etc.). Sustained release oral delivery systems and / or enteric coatings for oral dosage forms are described in U.S. Patent Nos. 4,704,295; U.S. Patent No. 4,556,552; U.S. Patent No. 4,309,406; And US Patent No. 4,309,404. Adjuvants are included under this passage.

The oligonucleotides and / or pharmaceutical compositions according to the present invention, in combination with those used in methods of preventing and / or treating human diseases, may also be used in veterinary animals, reptiles, birds, foreign animals and farm animals, such as mammals, rodents, etc. Used in the prevention and / or treatment of other patients, including. Mammals include, for example, horses, dogs, pigs, cats, or primates (eg monkeys, chimpanzees, or lemurs). Rodents include, for example, rats, rabbits, mice, squirrels or guinea pigs.

The oligonucleotide or pharmaceutical composition according to the invention can be used in a number of different diseases, preferably malignant or benign tumors, immunological diseases, bronchial asthma, heart disease, fibrosis (e.g. liver fibrosis, idiopathic pulmonary fibrosis, cirrhosis of the kidney, renal sclerosis, Scleroderma), diabetes, wound healing, diseases of connective tissue (e.g. heart, blood vessels, bones, joints, eyes, e.g. Marfan or Roy-Diet's syndrome), psoriasis, eye disease (e.g. glaucoma, posterior capsule turbidity) (PCO), retinoblastoma, choroidal carcinoma, macular degeneration, eg age-related macular degeneration, diabetic macular edema or cataracts), CNS disease (eg Alzheimer's disease, Parkinson's disease), coronary atherosclerosis (coronary artery) Used in interventional or coronary artery intervention or coronary artery bypass graft (CABG) or methods of prevention and / or treatment of alopecia. , Hemangioma, auditory neuroma, neurofibroma, trachoma, purulent granuloma, astrocytoma, for example retrograde astrocytoma, blastoma, Ewing's tumor, parietal papilloma, epithelioma, medulloblastoma, glioma, glioblastoma, angioblastoma, Hodgkin -Lymphoma, melanoma, e.g. primary and / or metastatic melanoma, mesothelioma, myeloma, neuroblastoma, neurofibroma, non-Hodgkin's lymphoma, pineal hematoma, retinoblastoma, sarcoma, normal hematoma, Wilm's tumor, cholangiocarcinoma, bladder carcinoma, Brain tumor, breast cancer, organ-supporting carcinoma, renal carcinoma, cervical cancer, chorionic cancer, cystic carcinoma, genital cancer, epithelial carcinoma, esophageal cancer, cervical carcinoma, colon carcinoma, colorectal carcinoma, endometrial cancer, gallbladder cancer, stomach cancer, head cancer, liver cancer , Lung carcinoma, medulla carcinoma, neck cancer, non-small cell organ support / lung carcinoma, ovarian cancer, pancreatic carcinoma, papillary carcinoma, papillary carcinoma, prostate cancer, small intestine carcinoma, prostate carcinoma, rectal cancer, renal cell carcinoma (RCC, e.g. Cell RCC, papillary RCC, achromoplastic RCC), eosinophilic cell kidney cancer, transitional cell kidney cancer, skin cancer, small cell organ support / lung carcinoma, squamous cell carcinoma, sebaceous carcinoma, testicular carcinoma, and uterine cancer. The oligonucleotide or pharmaceutical composition of is used not only for the method of preventing and / or treating tumors, but also for metastasis.

The invention is preferably an ophthalmic disease, such as but not limited to retinoblastoma, choroidal carcinoma, glaucoma, posterior capsule haze, dry eye, macular degeneration, eg age-related macular degeneration, diabetic macular edema, cataract, proliferative It relates to oligonucleotides for use in the methods of prevention and / or treatment of vitreoretinopathy, Marfan's or Roy-Diet's syndrome.

The antisense oligonucleotides of the present invention are characterized by exhibiting unexpectedly low toxicity and thus being well tolerated by different organisms. The oligonucleotide shows a suitable distribution in the organism, where the highest concentration is measured in the kidney, liver, skin and spleen.

The present invention provides a number of oligonucleotides that are highly efficient in reducing and inhibiting TGF-beta, in particular TGF-beta2 mRNA expression, respectively, due to specific selection of the sequence of the oligonucleotide and modification of the nucleotide. Table 1 below shows a number of preferred modified oligonucleotides according to the present invention (the bold letters indicate modified nucleosides). Each oligonucleotide is defined by ASPH and a number (limited by the specific sequence and modification of the nucleoside above):

[Table 1]

Table 1 shows the nucleic acid sequence of selected oligonucleotides of the present invention as well as modifications of the nucleotides, where LNA 4 + 4 means that 4 x LNA is modified at the 5'- and 3'-ends of the oligonucleotide, LNA 4 + 3 means that 4 x LNA at the 5'-end of the oligonucleotide and 3 x LNA at the 3'-end are modified, and LNA 3 + 4 is 3 x LNA at the 5'-end of the oligonucleotide and Means that 4 x LNA is modified at the 3'-end, LNA 3 + 3 means that 3 x LNA is modified at the 5'-end and 3'-end of the oligonucleotide, and LNA 3 + 2 is the oligonucleotide Means that 3 x LNA at the 5'-end of and 2 x LNA at the 3'-end are modified, LNA 2 + 3 is 2 x LNA at the 5'-end of the oligonucleotide and 3 x LNA at the 3'-end Means that is modified, LNA 2 + 2 is the 5'-end of the oligonucleotide Means that the 2 x LNA is modified at the 3'-end. On the one hand, some oligonucleotides are ENA 4 + 4 (i.e. 4 x ENA is modified at the 5'-end and 3'-end of the oligonucleotide) or ENA 3 + 3 (i.e. the 5'-end of the oligonucleotide and 3 'ENA at the 3'-end is modified). Additional oligonucleotides are 2'O-meth 4 + 4 (where the oligonucleotide includes 4 x 2'O-methyl modified nucleotides at the 5'-end and 3'-end of the oligonucleotide), or 2'fluoro 4 + 4 (where the oligonucleotide includes 4 x 2'fluoro modified nucleotides at the 5'-end and 3'-end). Oligonucleotides comprising LNA 3 + TEG include 3 x LNA at the 5'-end of the oligonucleotide and one triethylene glycol (TEG) at the 3'-end. Some oligonucleotides are not lined up, for example the sequences 1LNA + 1N + 1LNA + 8N + 3LNA, 3LNA + 8N + 1LNA + 1N + 1LNA, 3LNA + 8N + 1LNA + 1N + 2LNA, 3LNA + 8N + 2LNA + 1N + 1LNA, 2LNA + 1N + 1LNA + 8N + 3LNA, 1LNA + 1N + 2LNA + 8N + 3LNA, 1LNA + 2N + 2LNA + 8N + 3LNA, 1LNA + 3N + 1LNA + 8N + 3LNA, 1LNA + 2N + 2LNA + 8N + 4LNA, 1LNA + 2N + 2LNA + 8N + 1LNA + 1N + 2LNA, 1LNA + 1N + 3LNA + 8N + 3LNA, 1LNA + 1N + 2LNA + 8N + 3LNA, 1LNA + 2N + 3LNA + 8N + 2LNA, or 1LNA + 2N + 3LNA + Includes LNAs separated by unlocked (unmodified) nucleosides with 8N + 1LNA + 1N + 1LNA, where “N” is a nucleoside without a locking modification. In the above table, LNA nucleoside is represented by a sequence of dark letters, and triethylene glycol is abbreviated as TEG. “ASPH” in combination with a number refers to different oligonucleotides and their different modifications as disclosed in Table 1. The antisense oligonucleotides of the invention can be initiated differently, for example with ASPH47, ASPH0047, ASPH_47 or ASPH_0047 (referring to the same oligonucleotide). These modified oligonucleotides were tested, for example, in the experiments shown in the Examples below.

For purposes of clarity and conciseness, it will be appreciated that features are disclosed herein as part of the same or separate embodiments, but that the scope of the invention may include embodiments having a combination of some or all of the disclosed features. .

The following examples will be provided to further illustrate the invention, but at the same time, without configuring any limitations to the invention. On the contrary, it should be clearly understood that the scope of the present invention refers to various other embodiments, modifications, and equivalents thereof that may be implied to those skilled in the art without departing from the spirit of the present invention after reading the description of the present invention.

Example

In the following examples, the effects of the oligonucleotides listed in Table 1 were tested in light of reduction and inhibition of TGF-beta1 and / or TGF-beta2 expression, respectively. SEQ ID NO: 21 (T-LNA: CGGCATGTCTATTTTGTA, where the 3 x nucleotides at the 5'- and 3'-ends are LNA) and SEQ ID NO: 22 (scr-LNA: CGTTTAGGCTATGTACTT, where the 5'- and 3'-ends are 3 x nucleotide is LNA) as a control oligonucleotide, wherein SEQ ID NO: 22 (negative control) is the scrambled form of SEQ ID NO: 21 (positive control). The cells were transfected in the presence of a transfection agent (e.g. lipofectamine) or without any transfection agent (which is defined as a Jimnotic transfection or unsupported transfection or Jimnotic delivery). In the case of zymnotic delivery, entry of the oligonucleotide into the cell is entirely dependent on the interaction of the oligonucleotide with the cell (agonist does not support the entry). Therefore, it is believed that the gymnotic delivery reflects the better conditions of the in vivo environment.

Example 1

Human Panc-1 pancreatic cancer cells (FIG. 3A) or mouse RenCa renal cell carcinoma cells (FIG. 3B) in the absence of transfectants (Gymnotic transfection or Jimnotic delivery) 1.1 μM of ASPH190, ASPH191, ASPH192, ASPH193, respectively. ASPH194, ASPH195, ASPH196, ASPH197, ASPH198, ASPH199, ASPH200, ASPH201, ASPH202, ASPH203, ASPH204, ASPH205, ASPH206, ASPH207, ASPH208, ASPH209, ASPH210, ASPH211, ASPH212, ASPH213, ASPH214, ASPH215, ASPH216, ASPH217 It was treated with ASPH219, ASPH220, ASPH221, ASPH222, and ASPH223. Expression of TGF-beta 1 (black column), TGF-beta 2 (white column) and TGF-beta 3 (comb line column) mRNA was measured 72 hours after transfection. Significant reductions in the expression of TGF-beta2 mRNA are shown in FIGS. 3A and 3B. The negative control is scrambled LNA of SEQ ID NO: 22 (scr LNA).

Example 2

Human Panc-1 pancreatic cancer cells were treated with 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, and 0.12 μM ASPH47, M1-ASPH47, M2-ASPH47, respectively, in the absence of transfectants (Gymnotic transfection or Jimnotic delivery), respectively. M3-ASPH47, M4-ASPH47, M5-ASPH47, Μ6-ASPH47, M7-ASPH47, M8-ASPH47, M9-ASPH47, M10-ASPH47, M11-ASPH47, M12-ASPH47, M13-ASPH47, M14-ASPH47, or M15 -Treated with ASPH47. The inhibitory effect of the modified oligonucleotides on the expression of TGF-beta2 mRNA was measured 72 hours after the start of treatment. TGF-beta2 values were normalized to GAPDH and oligonucleotide concentrations (= IC ₅₀ ) resulting in a 50% reduction in TGF-beta2 mRNA were calculated. Under the conditions of the Jimnotic transfection experiment, the oligonucleotide enters the cell and strongly suppresses the expression of TGF-beta2 mRNA. Table 2 shows the results of the experiment:

[Table 2]

The modified oligonucleotides all exhibit IC ₅₀ in the submicromolar to lower submicromolar range, which indicates that the oligonucleotide has a very high potency even without the need for a transfection agent.

Example 3

Human Panc-1 pancreatic cancer cells were transfected with 20, 6.67, 2.22, 0.74, 0.25, 0.08 or 0.009 μM modified oligonucleotide ASPH47; The results are shown in Fig. 4A. The negative control is the scrambled oligonucleotide of SEQ ID NO: 22 (scrLNA) (Figure 4B). Cells were transfected in the absence of a transfectant (zymotic transfection or zymnotic delivery). The oligonucleotide was added to the cells for 3 days, and it was incubated at 37 ° C. The medium was then exchanged with fresh oligonucleotide-containing medium and the cells were incubated for an additional 4 days at 37 ° C. TGF-beta1 and TGF-beta2 protein levels in cell supernatant were measured by ELISA. ASPH47 specifically inhibits the expression of TGF-beta2 in a dose-dependent manner and does not show a target inhibitory effect on TGF-beta1 (FIG. 4A). The scrLNA of SEQ ID NO: 22, during doubling the concentration compared to the individual concentration of ASPH47 (40, 13.33, 4.44, 1.48, 0.49, 0.16, 0.05, or 0.02 μΜ), during expression of TGF-beta1 or TGF-beta2 It does not show any inhibitory effect on either. The results for TGF-beta 1 are represented by diamonds, and the results for TGF-beta 2 are represented by squares (FIGS. 4A to 4B).

                         SEQUENCE LISTING <110> ISARNA Therapeutics GmbH   <120> Modified TGF-beta2 oligonucleotides <130> P101116PC20 <140> PCT / EP2014 / 056232 <141> 2014-03-27 <150> EP 13161474.5 <151> 2013-03-27 <150> EP 13173078.0 <151> 2013-06-20 <150> EP 13199838.7 <151> 2013-12-30 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 5882 <212> DNA <213> Homo sapiens <400> 1 gtgatgttat ctgctggcag cagaaggttc gctccgagcg gagctccaga agctcctgac 60 aagagaaaga cagattgaga tagagataga aagagaaaga gagaaagaga cagcagagcg 120 agagcgcaag tgaaagaggc aggggagggg gatggagaat attagcctga cggtctaggg 180 agtcatccag gaacaaactg aggggctgcc cggctgcaga caggaggaga cagagaggat 240 ctattttagg gtggcaagtg cctacctacc ctaagcgagc aattccacgt tggggagaag 300 ccagcagagg ttgggaaagg gtgggagtcc aagggagccc ctgcgcaacc ccctcaggaa 360 taaaactccc cagccagggt gtcgcaaggg ctgccgttgt gatccgcagg gggtgaacgc 420 aaccgcgacg gctgatcgtc tgtggctggg ttggcgtttg gagcaagaga aggaggagca 480 ggagaaggag ggagctggag gctggaagcg tttgcaagcg gcggcggcag caacgtggag 540 taaccaagcg ggtcagcgcg cgcccgccag ggtgtaggcc acggagcgca gctcccagag 600 caggatccgc gccgcctcag cagcctctgc ggcccctgcg gcacccgacc gagtaccgag 660 cgccctgcga agcgcaccct cctccccgcg gtgcgctggg ctcgccccca gcgcgcgcac 720 acgcacacac acacacacac acacacacgc acgcacacac gtgtgcgctt ctctgctccg 780 gagctgctgc tgctcctgct ctcagcgccg cagtggaagg caggaccgaa ccgctccttc 840 tttaaatata taaatttcag cccaggtcag cctcggcggc ccccctcacc gcgctcccgg 900 cgcccctccc gtcagttcgc cagctgccag ccccgggacc ttttcatctc ttcccttttg 960 gccggaggag ccgagttcag atccgccact ccgcacccga gactgacaca ctgaactcca 1020 cttcctcctc ttaaatttat ttctacttaa tagccactcg tctctttttt tccccatctc 1080 attgctccaa gaattttttt cttcttactc gccaaagtca gggttccctc tgcccgtccc 1140 gtattaatat ttccactttt ggaactactg gccttttctt tttaaaggaa ttcaagcagg 1200 atacgttttt ctgttgggca ttgactagat tgtttgcaaa agtttcgcat caaaaacaac 1260 aacaacaaaa aaccaaacaa ctctccttga tctatacttt gagaattgtt gatttctttt 1320 ttttattctg acttttaaaa acaacttttt tttccacttt tttaaaaaat gcactactgt 1380 gtgctgagcg cttttctgat cctgcatctg gtcacggtcg cgctcagcct gtctacctgc 1440 agcacactcg atatggacca gttcatgcgc aagaggatcg aggcgatccg cgggcagatc 1500 ctgagcaagc tgaagctcac cagtccccca gaagactatc ctgagcccga ggaagtcccc 1560 ccggaggtga tttccatcta caacagcacc agggacttgc tccaggagaa ggcgagccgg 1620 agggcggccg cctgcgagcg cgagaggagc gacgaagagt actacgccaa ggaggtttac 1680 aaaatagaca tgccgccctt cttcccctcc gaaaatgcca tcccgcccac tttctacaga 1740 ccctacttca gaattgttcg atttgacgtc tcagcaatgg agaagaatgc ttccaatttg 1800 gtgaaagcag agttcagagt ctttcgtttg cagaacccaa aagccagagt gcctgaacaa 1860 cggattgagc tatatcagat tctcaagtcc aaagatttaa catctccaac ccagcgctac 1920 atcgacagca aagttgtgaa aacaagagca gaaggcgaat ggctctcctt cgatgtaact 1980 gatgctgttc atgaatggct tcaccataaa gacaggaacc tgggatttaa aataagctta 2040 cactgtccct gctgcacttt tgtaccatct aataattaca tcatcccaaa taaaagtgaa 2100 gaactagaag caagatttgc aggtattgat ggcacctcca catataccag tggtgatcag 2160 aaaactataa agtccactag gaaaaaaaac agtgggaaga ccccacatct cctgctaatg 2220 ttattgccct cctacagact tgagtcacaa cagaccaacc ggcggaagaa gcgtgctttg 2280 gatgcggcct attgctttag aaatgtgcag gataattgct gcctacgtcc actttacatt 2340 gatttcaaga gggatctagg gtggaaatgg atacacgaac ccaaagggta caatgccaac 2400 ttctgtgctg gagcatgccc gtatttatgg agttcagaca ctcagcacag cagggtcctg 2460 agcttatata ataccataaa tccagaagca tctgcttctc cttgctgcgt gtcccaagat 2520 ttagaacctc taaccattct ctactacatt ggcaaaacac ccaagattga acagctttct 2580 aatatgattg taaagtcttg caaatgcagc taaaattctt ggaaaagtgg caagaccaaa 2640 atgacaatga tgatgataat gatgatgacg acgacaacga tgatgcttgt aacaagaaaa 2700 cataagagag ccttggttca tcagtgttaa aaaatttttg aaaaggcggt actagttcag 2760 acactttgga agtttgtgtt ctgtttgtta aaactggcat ctgacacaaa aaaagttgaa 2820 ggccttattc tacatttcac ctactttgta agtgagagag acaagaagca aatttttttt 2880 aaagaaaaaa ataaacactg gaagaattta ttagtgttaa ttatgtgaac aacgacaaca 2940 acaacaacaa caacaaacag gaaaatccca ttaagtggag ttgctgtacg taccgttcct 3000 atcccgcgcc tcacttgatt tttctgtatt gctatgcaat aggcaccctt cccattctta 3060 ctcttagagt taacagtgag ttatttattg tgtgttacta tataatgaac gtttcattgc 3120 ccttggaaaa taaaacaggt gtataaagtg gagaccaaat actttgccag aaactcatgg 3180 atggcttaag gaacttgaac tcaaacgagc cagaaaaaaa gaggtcatat taatgggatg 3240 aaaacccaag tgagttatta tatgaccgag aaagtctgca ttaagataaa gaccctgaaa 3300 acacatgtta tgtatcagct gcctaaggaa gcttcttgta aggtccaaaa actaaaaaga 3360 ctgttaataa aagaaacttt cagtcagaat aagtctgtaa gttttttttt ttctttttaa 3420 ttgtaaatgg ttctttgtca gtttagtaaa ccagtgaaat gttgaaatgt tttgacatgt 3480 actggtcaaa cttcagacct taaaatattg ctgtatagct atgctatagg ttttttcctt 3540 tgttttggta tatgtaacca tacctatatt attaaaatag atggatatag aagccagcat 3600 aattgaaaac acatctgcag atctcttttg caaactatta aatcaaaaca ttaactactt 3660 tatgtgtaat gtgtaaattt ttaccatatt ttttatattc tgtaataatg tcaactatga 3720 tttagattga cttaaatttg ggctcttttt aatgatcact cacaaatgta tgtttctttt 3780 agctggccag tacttttgag taaagcccct atagtttgac ttgcactaca aatgcatttt 3840 ttttttaata acatttgccc tacttgtgct ttgtgtttct ttcattatta tgacataagc 3900 tacctgggtc cacttgtctt ttcttttttt tgtttcacag aaaagatggg ttcgagttca 3960 gtggtcttca tcttccaagc atcattacta accaagtcag acgttaacaa atttttatgt 4020 taggaaaagg aggaatgtta tagatacata gaaaattgaa gtaaaatgtt ttcattttag 4080 caaggattta gggttctaac taaaactcag aatctttatt gagttaagaa aagtttctct 4140 accttggttt aatcaatatt tttgtaaaat cctattgtta ttacaaagag gacacttcat 4200 aggaaacatc tttttcttta gtcaggtttt taatattcag ggggaaattg aaagatatat 4260 attttagtcg atttttcaaa aggggaaaaa agtccaggtc agcataagtc attttgtgta 4320 tttcactgaa gttataaggt ttttataaat gttctttgaa ggggaaaagg cacaagccaa 4380 tttttcctat gatcaaaaaa ttctttcttt cctctgagtg agagttatct atatctgagg 4440 ctaaagttta ccttgcttta ataaataatt tgccacatca ttgcagaaga ggtatcctca 4500 tgctggggtt aatagaatat gtcagtttat cacttgtcgc ttatttagct ttaaaataaa 4560 aattaatagg caaagcaatg gaatatttgc agtttcacct aaagagcagc ataaggaggc 4620 gggaatccaa agtgaagttg tttgatatgg tctacttctt ttttggaatt tcctgaccat 4680 taattaaaga attggatttg caagtttgaa aactggaaaa gcaagagatg ggatgccata 4740 atagtaaaca gcccttgtgt tggatgtaac ccaatcccag atttgagtgt gtgttgatta 4800 tttttttgtc ttccactttt ctattatgtg taaatcactt ttatttctgc agacattttc 4860 ctctcagata ggatgacatt ttgttttgta ttattttgtc tttcctcatg aatgcactga 4920 taatatttta aatgctctat tttaagatct cttgaatctg tttttttttt ttttaatttg 4980 ggggttctgt aaggtcttta tttcccataa gtaaatattg ccatgggagg ggggtggagg 5040 tggcaaggaa ggggtgaagt gctagtatgc aagtgggcag caattatttt tgtgttaatc 5100 agcagtacaa tttgatcgtt ggcatggtta aaaaatggaa tataagatta gctgttttgt 5160 attttgatga ccaattacgc tgtattttaa cacgatgtat gtctgttttt gtggtgctct 5220 agtggtaaat aaattatttc gatgatatgt ggatgtcttt ttcctatcag taccatcatc 5280 gagtctagaa aacacctgtg atgcaataag actatctcaa gctggaaaag tcataccacc 5340 tttccgattg ccctctgtgc tttctccctt aaggacagtc acttcagaag tcatgcttta 5400 aagcacaaga gtcaggccat atccatcaag gatagaagaa atccctgtgc cgtcttttta 5460 ttcccttatt tattgctatt tggtaattgt ttgagattta gtttccatcc agcttgactg 5520 ccgaccagaa aaaatgcaga gagatgtttg caccatgctt tggctttctg gttctatgtt 5580 ctgccaacgc cagggccaaa agaactggtc tagacagtat cccctgtagc cccataactt 5640 ggatagttgc tgagccagcc agatataaca agagccacgt gctttctggg gttggttgtt 5700 tgggatcagc tacttgcctg tcagtttcac tggtaccact gcaccacaaa caaaaaaacc 5760 caccctattt cctccaattt ttttggctgc tacctacaag accagactcc tcaaacgagt 5820 tgccaatctc ttaataaata ggattaataa aaaaagtaat tgtgactcaa aaaaaaaaaa 5880 aa 5882 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 2 caaagtattt ggtctcc 17 <210> 3 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 3 agtatttggt ctcc 14 <210> 4 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 4 aagtatttgg tctc 14 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 5 aagtatttgg tctcc 15 <210> 6 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 6 agtatttggc tctc 14 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 7 agtatttggt ctcca 15 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 8 aagtatttgg tctcc 15 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 9 caaagtattt ggtctc 16 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 10 caaagtattt ggtct 15 <210> 11 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 11 caaagtattt ggtc 14 <210> 12 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 12 aaagtatttg gtctcc 16 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 13 aaagtatttg gtctc 15 <210> 14 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 14 aaagtatttg gtct 14 <210> 15 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 15 aaagtatttg gtc 13 <210> 16 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 16 aagtatttgg tct 13 <210> 17 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 17 aagtatttgg tc 12 <210> 18 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 18 agtatttggt ctc 13 <210> 19 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 19 agtatttggt ct 12 <210> 20 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> modified TGF-beta2 oligonucleotide <400> 20 agtatttggt c 11

Claims (8)

SEQ ID NO: 2 (CAAAGTATTTGGTCTCC), SEQ ID NO: 3 (AGTATTTGGTCTCC), SEQ ID NO: 4 (AAGTATTTGGTCTC), SEQ ID NO: 5 (AAGTATTTGGTCTCC), SEQ ID NO: 6 (AGTATTTGGTCTCC), SEQ ID NO: 7 (AGTATTTGGTCTCCA), SEQ ID NO: 8 (AAGTATTTGGTCTCC) Number 9 (CAAAGTATTTGGTCTC), SEQ ID NO: 10 (CAAAGTATTTGGTCT), SEQ ID NO: 11 (CAAAGTATTTGGTCT), SEQ ID NO: 12 (AAAGTATTTGGTCTCC), SEQ ID NO: 13 (AAAGTATTTGGTCTC), SEQ ID NO: 14 (AAAGTATTTGGTCT), SEQ ID NO: 15 (AAAGTATTTGGTC), SEQ ID NO: 15 Antisense oligonucleotide consisting of a sequence selected from the group consisting of 16 (AAGTATTTGGTCT), SEQ ID NO: 17 (AAGTATTTGGTC), SEQ ID NO: 18 (AGTATTTGGTCTC), SEQ ID NO: 19 (AGTATTTGGTCT) and SEQ ID NO: 20 (AGTATTTGGTC),
One or more nucleotide (s) of the oligonucleotide is modified, and the modified nucleotides are LNA, ENA, polyalkylene oxide-, 2'-fluoro-, 2'-O-methoxy-, and 2'-O At least one selected from the group consisting of -methyl-modified nucleotides,
An antisense oligonucleotide wherein the modified nucleotide is located at the 5'-end or 3'-end or 5'-end and 3'-end of the oligonucleotide. delete According to claim 1,
Oligonucleotide SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and antisense oligonucleotide consisting of a sequence selected from the group consisting of SEQ ID NO: 18. According to claim 1,
Oligonucleotides are AGT ATTTGGTC TCC (SEQ ID NO: 3), AAG TATTTGGT CTC (SEQ ID NO: 4), AAG TATTTGGTC TCC (SEQ ID NO: 5), CAAA GTATTTGGT CTCC (SEQ ID NO: 2), AG TATTTGGTC TCC (SEQ ID NO: 6), A G T ATTTGGTCT CC (SEQ ID NO: 6), AGT ATTTGGTC T C C (SEQ ID NO: 6), AGT ATTTGGTCT CC (SEQ ID NO: 6), AAGT ATTTGGTC TC (SEQ ID NO: 4), AGT ATTTGGTC T C CA (SEQ ID NO: 7), AGT ATTTGGTC TC C A (SEQ ID NO: 7), AG T A TTTGGTCT CCA (SEQ ID NO: 7), A G TA TTTGGTCT CCA (SEQ ID NO: 7), AGT ATTTGGTCTC CA (SEQ ID NO: 7), AG TATTTGGTCT CCA (SEQ ID NO: 7), AG TATTTGGTC TCCA (SEQ ID NO: 7), AAG TATTTGGT C T CC (SEQ ID NO: 8), AAG TATTTGGT CT C C (SEQ ID NO: 8), AA G T ATTTGGTC TCC (SEQ ID NO: 8), A A GT ATTTGGTC TCC (SEQ ID NO: 8), AAG TATTTGGTCT CC (SEQ ID NO: 8), AA GTATTTGGTC TCC (SEQ ID NO: 8), CAA AGTATTTGGTC TCC (SEQ ID NO: 2), CA AAGTATTTGGTCT CC (SEQ ID NO: 2), C AA AG TATTTGGTC TCC (SEQ ID NO: 2), C AAA G TATTTGGTC TCC (SEQ ID NO: 2), C A A AG TATTTGGT CTCC (SEQ ID NO: 2), C AA AG TATTTGGT C T CC (SEQ ID NO: 2), C A AAG TATTTGGTC TCC (SEQ ID NO: 2), C A AA GTATTTGGTC TCC (SEQ ID NO: 2), C AA AGT ATTTGGTCT CC (SEQ ID NO: 2), C AA AGT ATTTGGTC T C C (SEQ ID NO: 2), CAAA GTATTTGGT CTC (SEQ ID NO: 9), CAAA GTATTTGGT CT (SEQ ID NO: 10), CAAA GTATTTGGT C (SEQ ID NO: 11), AAA GTATTTGGT CTCC ( SEQ ID NO: 12), AAA GTATTTGGT CTC (SEQ ID NO: 13), AAA GTATTTGGT CT (SEQ ID NO: 14), AAA GTATTTGGT C (SEQ ID NO: 15), AAG TATTTGGTC TCC (SEQ ID NO: 5), AAG TATTTGGT CTC (SEQ ID NO: 4), AA GTATTTGGT CT (SEQ ID NO: 16), AA GTATTTGGT C (SEQ ID NO: 17), AGT ATTTGGTC TCC (SEQ ID NO: 3), A GTATTTGGT CTC (SEQ ID NO: 18), A GTATTTGGT CT (SEQ ID NO: 19) and A GTATTTGGT C (SEQ ID NO: No. 20) is a sequence selected from the group consisting of, wherein the nucleotide described in bold is LNA modified nucleotide, antisense oligonucleotide. The antisense oligonucleotide according to claim 1 and optionally comprising a pharmaceutically acceptable carrier, a method for preventing or treating or preventing or treating a disease selected from malignant tumors, benign tumors, immunological diseases, fibrosis and ophthalmic diseases As a pharmaceutical composition for use in,
The immunological disease is associated with TGF-beta2 overexpression,
The ophthalmic disease is a pharmaceutical composition selected from the group consisting of glaucoma, posterior capsule turbidity, dry eye, macular degeneration, diabetic macular edema, cataract, proliferative vitreoretinopathy and Marfan and Roy-Diet's syndrome. According to claim 1,
As an antisense oligonucleotide for use in a method or method of preventing or treating a disease selected from malignant tumors, benign tumors, immunological diseases, fibrosis and ophthalmic diseases,
The immunological disease is associated with TGF-beta2 overexpression,
The ophthalmic disease is an antisense oligonucleotide selected from the group consisting of glaucoma, posterior capsule opacity, dry eye, macular degeneration, diabetic macular edema, cataract, proliferative vitreoretinopathy and marfan and Roy-Diet's syndrome. The method of claim 6,
Tumors are solid tumors, hematologic tumors, leukemias, tumor metastases, hemangioma, auditory neomas, neurofibromas, trachoma, purulent granulomas, psoriasis, astrocytoma, blastoma, Ewing's tumor, parietal head tumor, stromal cell tumor, medulloblastoma, glioma, hemangioma Selected from the group consisting of blastoma, Hodgkin-lymphoma, mesothelioma, neuroblastoma, non-Hodgkin's lymphoma, cryptoma, retinoblastoma, sarcoma, normal hematoma, and Wilm's tumor, or bile duct cancer, bladder carcinoma, brain tumor, breast cancer, and organ-supporting carcinoma , Kidney carcinoma, cervical cancer, chorionic cancer, cystic carcinoma, genital carcinoma, epithelial carcinoma, esophageal cancer, cervical carcinoma, colon carcinoma, colorectal carcinoma, endometrial cancer, gallbladder cancer, stomach cancer, head cancer, liver carcinoma, lung carcinoma, medulla carcinoma, Neck cancer, non-small cell organ support / lung carcinoma, ovarian cancer, pancreatic carcinoma, papillary carcinoma, papillary carcinoma, prostate cancer, small intestine carcinoma, prostate carcinoma, rectal cancer, renal cell carcinoma, skin cancer, small cell organ support / lung Antisense oligonucleotide selected from the group consisting of carcinoma, squamous cell carcinoma, sebaceous carcinoma, testicular carcinoma, and uterine cancer. The method of claim 5,
Tumors are solid tumors, hematologic tumors, leukemias, tumor metastases, hemangioma, auditory neomas, neurofibromas, trachoma, purulent granulomas, psoriasis, astrocytoma, blastoma, Ewing's tumor, parietal head tumor, stromal cell tumor, medulloblastoma, glioma, hemangioma Selected from the group consisting of blastoma, Hodgkin-lymphoma, mesothelioma, neuroblastoma, non-Hodgkin's lymphoma, cryptoma, retinoblastoma, sarcoma, normal hematoma, and Wilm's tumor, or bile duct cancer, bladder carcinoma, brain tumor, breast cancer, and organ-supporting carcinoma , Renal carcinoma, cervical cancer, chorionic cancer, cystic carcinoma, genital carcinoma, epithelial carcinoma, esophageal cancer, cervical carcinoma, colon carcinoma, colorectal carcinoma, endometrial cancer, gallbladder cancer, stomach cancer, head cancer, hepatocarcinoma, lung carcinoma, medulla carcinoma, Neck cancer, non-small cell organ support / lung carcinoma, ovarian cancer, pancreatic carcinoma, papillary carcinoma, papillary carcinoma, prostate cancer, small intestine carcinoma, prostate carcinoma, rectal cancer, renal cell carcinoma, skin cancer, small cell organ support / lung A pharmaceutical composition selected from the group consisting of carcinoma, squamous cell carcinoma, sebaceous carcinoma, testicular carcinoma, and uterine cancer.

Images